Pcdna3 manual

DNA yield can be assessed using three different physical methods: absorbance optical density , agarose gel electrophoresis and fluorescent DNA-binding dyes.

Each technique is described below and includes information on necessary accessories e. While all methods are useful, each has caveats to consider when choosing a quantitation approach. The most common technique to determine DNA yield and purity is also the easiest method—absorbance. All that is needed for measurement is a spectrophotometer equipped with a UV lamp, UV-transparent cuvettes depending on the instrument and a solution of purified DNA.

Absorbance readings are performed at nm A where DNA absorbs light most strongly, and the number generated allows one to estimate the concentration of the solution. To ensure the numbers are useful, the A reading should be between 0. Since RNA also has a great absorbance at nm, and the aromatic amino acids present in protein absorb at nm, both contaminants, if present in the DNA solution, will contribute to the total measurement at nm.

Additionally, the presence of guanidine will lead to higher nm absorbance. This means that if the A number is used for calculation of yield, the DNA quantity may be overestimated To evaluate DNA purity by spectrophotometry, measure absorbance from nm to nm in order to detect other possible contaminants present in the DNA solution.

The most common purity calculation is determining the ratio of the absorbance at nm divided by the reading at nm. A reading of 1. However, the best test of DNA quality is functionality in the application of interest e.

Strong absorbance around nm can indicate that organic compounds or chaotropic salts are present in the purified DNA. A ratio of nm to nm can help evaluate the level of salt carryover in the purified DNA. The lower the ratio, the greater the amount of thiocyanate salt is present, for example. A reading at nm will indicate if there is turbidity in the solution, another indication of possible contamination.

Therefore, taking a spectrum of readings from nm to nm is most informative. Agarose gel electrophoresis of the purified DNA eliminates some of the issues associated with absorbance readings. To use this method, a horizontal gel electrophoresis tank with an external power supply, analytical-grade agarose, an appropriate running buffer e.

A sample of the isolated DNA is loaded into a well of the agarose gel and then exposed to an electric field. The negatively charged DNA backbone migrates toward the anode. The percentage of agarose in the gel will determine what size range of DNA will be resolved with the greatest clarity Concentration and yield can be determined after gel electrophoresis is completed by comparing the sample DNA intensity to that of a DNA quantitation standard.

Standards used for quantitation should be labeled as such and be the same size as the sample DNA being analyzed. Because ethidium bromide is a known mutagen, precautions need to be taken for its proper use and disposal DNA-binding dyes compare the unknown sample to a standard curve of DNA, but genomic, fragment and plasmid DNA will each require their own standard curves and cannot be used interchangeably.

If the DNA sample has been diluted, you will need to account for the dilution factor when calculating final concentration. To use this method, a fluorometer to detect the dyes, dilution of the DNA solution and appropriate DNA standards are required. In addition, the usual caveats for handling fluorescent compounds apply—photobleaching and quenching will affect the signal.

Choosing which quantitation method to use is based on many factors including access to equipment or reagents, reliability and consistency of the concentration calculations. Use caution when comparing yields between methods as the level of potential contaminants may cause variable determinations among the different methods.

In this DNA purification guide, we discussed the basic steps of DNA extraction, plasmid preparation and DNA quantitation, and explored the vast portfolio of products that Promega has to offer.

This guide is intended to help you understand those basics, navigate issues of scalability, purity, yield and the effects they have on downstream applications, and ultimately assist you in identifying the system that best fits your DNA purification needs.

Need additional assistance? Here at Promega, your success is important to us and we genuinely enjoy the challenge of identifying the right product to address your technical needs. Our Technical Services department is available to help guide you every step of the way, from answering technical questions about your products to providing support for your automated instruments. We use these cookies to ensure our site functions securely and properly; they are necessary for our services to function and cannot be switched off in our systems.

They are usually only set in response to actions made by you which amount to a request for services, such as logging in, using a shopping cart or filling in forms. You can set your browser to block or alert you about these cookies, but some parts of our services will not work without them.

Like the other cookies we use, strictly necessary cookies may be either first-party cookies or third - party cookies. We use these cookies to remember your settings and preferences. For example, we may use these cookies to remember your language preferences. We use these cookies to collect information about how you interact with our services and to help us measure and improve them. For example, we may use these cookies to determine if you have interacted with a certain page.

We and our advertising partners use these cookies to deliver advertisements, to make them more relevant and meaningful to you, and to track the efficiency of our advertising campaigns, both on our services and on other websites and social media.

Promega's Cookie Policy We use cookies and similar technologies to make our website work, run analytics, improve our website, and show you personalized content and advertising. Yes, I Accept. Your Account Username Account not found. Email address is unverified. Account is locked. Password Incorrect password. Password reset is required. Account is invalid.

Log In. Create an Account. Contact Customer Service Forgot Password? To protect your privacy, your account has been locked after 6 failed login attempts. Please contact Customer Service to unlock your account. Enter your username and we'll send a link to reset your password. Username Username not found. Send Email. A password reset email has been sent to the primary email address associated with your account.

Current Password Incorrect password. New Password Minimum of 8 characters Uppercase and lowercase letters At least one number. Password doesn't meet requirements. Password has been used too recently. Confirm New Password Passwords don't match. You have successfully reset your password. Your password reset link has expired. Please request another reset link. Reset Password. There was an issue resetting your password. There was an issue logging into your account.

A verification email has been sent to the primary email address associated with your account. There was an issue sending the verification email. Name This field is required. Email Address Please enter a valid email address. Username already in use. Password Minimum of 8 characters Uppercase and lowercase letters At least one number. Confirm Password Passwords don't match.

Create Account. By creating an account, you confirm that you accept the Terms and Conditions and Privacy Policy. Have an account? You've created a Promega. Check your inbox to complete email verification. Note: You will not be able to access your account until your email is verified. Our records indicate that this email address is already registered. There was an issue creating your account.

There was an error processing your request. Please check your network settings and try again. Close You've successfully logged out. Thank you for verifying your email address. There was an issue verifying your email address. Global Support Our customer and technical support experts are here to help!

Explore Support Center Medical Affairs We provide medical information and facilitate research collaborations. Connect With Us. Local Sales Support Get in touch with a nearby distributor or sales representative. Find Sales Contact.

Contact Us Customer Support. Learn How. Go to Products. DNA Purification Basics Basic Isolation Procedure There are five basic steps of DNA extraction that are consistent across all the possible DNA purification chemistries: 1 disruption of the cellular structure to create a lysate, 2 separation of the soluble DNA from cell debris and other insoluble material, 3 binding the DNA of interest to a purification matrix, 4 washing proteins and other contaminants away from the matrix and 5 elution of the DNA.

Physical Methods Physical methods typically involve some type of sample grinding or crushing to disrupt the cell walls or tough tissue. Chemical Methods Chemical methods can be used alone with easy-to-lyse materials, such as tissue culture cells or in combination with other methods. Enzymatic Methods Enzymatic methods are often used with more structured starting materials in combination with other methods with tissues, plant materials, bacteria and yeast. Clearing of Lysate Depending on the starting material, cellular lysates may need to have cellular debris removed prior to nucleic acid purification to reduce the carryover of unwanted materials proteins, lipids and saccharides from cellular structures into the purification reaction, which can clog membranes or interfere with downstream applications.

Binding to the Purification Matrix Regardless of the method used to create a cleared lysate, the DNA of interest can be isolated using a variety of different methods. Solution-Based Chemistry This type of chemistry does not rely on a binding matrix, but rather on alcohol precipitation. Figure 1. Images of two Promega silica purification matrices. The membrane is present at the bottom of the column. Ion Exchange Chemistry Ion exchange chemistry is based on the interaction that occurs between positively-charged particles and the negatively-charged phosphates that are present in DNA.

Washing Wash buffers generally contain alcohols and can be used to remove proteins, salts and other contaminants from the sample or the upstream binding buffers. Manual Purification Systems Solution-Based Systems Promega offers genomic DNA isolation systems based on sample lysis by detergents and purification by various methods. Automated Systems for DNA Purification As laboratories try to improve productivity for research, diagnostics and applied testing, the need has increased for easy-to-use, low- to moderate-throughput automation of purification processes.

Cartridge-Based Systems Traditionally, automation refers to the use of large, specialized and costly equipment that requires extensive training to operate and maintain. Figure 7. Custom HT Nucleic Acid Purification Implementing automated nucleic acid purification technologies onto your high-throughput workflow can be challenging and time-consuming. Selected DNA Purification Kits by Sample Type Learn more about some of our specialized kits below, and explore the breadth of our portfolio and compare our DNA extraction kits with the help of our product comparison page to discover the right solution for your DNA purification needs.

NGS is another assay used by some labs to QC their samples. There are several reasons for this. Some labs are trying to get as much data as possible from very precious samples, in which case any sequence information may be worth the expense and risk of failed sequencing runs. As a QC test, NGS may provide a lot of information, but it is expensive and can require large amounts of sample and time. Some labs run low pass NGS, which uses highly-multiplexed samples to lower the cost per sample to determine if it is worth the time and resources to sequence deeper.

Table 4. Method Speed Sensitivity Quantitative? Measure Purity? Assess size of NA? Detect Cross- linking? Food Genomic DNA Isolation Food and plant materials often provide the greatest challenge for cell lysis and intact DNA extraction, due to the lysis conditions required to liberate the nucleic acid and the processing of plant materials into comestibles.

Depending on inoculation size and the size of the culture, stationary phase will be reached in 6—8 hours Aeration and temperature are of critical importance. The culture tube or flask should be placed in an orbital shaker approximately rpm to ensure adequate aeration Baffled flasks may increase aeration and thus yields of plasmid DNA. Since most strains of E. Antibiotic Selection Most plasmids carry a marker gene for a specific antibiotic resistance. Table 5.

Antibiotic Mode of Action and Mechanism of Resistance. Stock Solution Ampicillin Amp A derivative of penicillin that kills growing cells by interfering with bacterial cell wall synthesis.

The resistance gene cat specifies an acetyltransferase that acetylates, and thereby inactivates, the antibiotic. The resistance gene kan specifies an enzyme aminoglycoside phosphotransferase that modifies the antibiotic and prevents its interaction with ribosomes. Expression of the bacterial APH aminoglycoside phosphotransferase gene derived from Tn5.

The resistance gene tet specifies a protein that modifies the bacterial membrane and prevents transport of the antibiotic into the cell. Recommended Inoculation Procedures 1—ml of Culture Pick an isolated colony from a freshly streaked plate less than 5 days old and inoculate LB medium containing the required antibiotic s. Plasmid Copy Number One of the most critical factors affecting the yield of plasmid from a given system is the copy number of the plasmid.

Appropriate Sample Size and Throughput Depending on the volume of the bacterial culture, there are different isolation systems for your needs.

Biomass Processed Optical density O. Plasmid Purification Method and Transfection Many plasmid isolation systems indicate they are transfection-quality e. An additional benefit is that the same degree of purification can be obtained even with low-copy-number plasmids.

Although the system works best for plasmids less than 10kb, plasmids as large as 18kb have been purified. References Mandrekar, P. Chen, C. Marko, M. Boom, R. Melzak, K. Colloid Interface Sci. USA , — Walker, J. Zhang, X. Plant J. Flashner, Y. Lee, P. Martinez, C. Ahmed, A. Pereira, C. Cox, A. Sports Exerc. Lorenzen, M. Application Notes. Stump, A. Dontu, G. Genes Dev. Park, J. Teresa Pellicer, M. Smith, P. Ohsaki, E. Birnboim, H. Acids Res. Methods Enzymol.

Hirt, B. Holmes, D. Hamaguchi, K. Wilcockson, J. Lis, J. Paithankar, K. Nucleic Acids Res. Wang, Z. Ausubel, F. Davies, J. Lehman, I. Purification and properties of a ribonucleic acid-inhibitable endonuclease. Goebel, W. Transfer ribonucleic acid complex of Escherichia coli. Shortman, K. Changes in enzyme levels in response to alterations in physiological state. Schoenfeld, T. Promega Notes. Sambrook, J.

Lewin, B. Butash, K. Adams, D. Some of these regions of differences which encode antigens are present in the genome of M. MPT64 is one of the best-characterized antigens in RD2 the second absent region from the original BCG strains and a major secreted protein in the early culture filtrate of M.

This protein consists of amino acids with molecular weight of DNA extraction was performed using boiling method The mpt64 gene amplification was performed using the following primers, forward:. Both pcDNA3. After ligation, the competent cells of Escherichia coli E. The prepared E. This step also included positive and negative controls for validating the process of competent cell preparation. Colonies acquiring the recombinant vector were confirmed using Colony-PCR.

Germany and the results were analyzed by BioEdit software. In this study, we amplified M. After gel purification of PCR product, single and double digestions were performed on the product. The pcDNA3. Then, mpt64 fragment was ligated into the pcDNA3. The ligation product was used to transform E. Colony-PCR was performed using mpt64 specific primers to confirm insertion of our fragment into the vector and the bp fragment was observed by gel electrophoresis.

Then the recombinant vector was subjected to double digestion with Bam HI and Eco RI, so the insert cut out and was observed by gel electrophoresis. Final confirmation was based on DNA sequencing of recombinant vector. The long duration of conventional chemotherapy of TB, together with the increasing incidence of drug-resistant strains and co-infection with HIV, points to an urgent need for new strategies against TB BCG is the only available vaccine and a live attenuated one which has been used since Despite the worldwide use, it has some limitations such as low level of protection in pulmonary TB.

So, TB remains as the major public health problem 18 , Thus, new effective and reliable vaccines are needed. In this regard, several strategies including DNA vaccines, recombinant BCG vaccines, and subunit vaccines are being developed. The secreted and cell wall proteins of M.

Mahairas and colleagues demonstrated the M. Their studies showed that three specific regions in the genome of M. MPT64 Rvc , an important immunodominant antigen with superoxide dismutase activity and a strong cellular immune activity, is encoded by RD2 of M.

This protein has amino acids with the molecular weight of According to their research, the use of DNA vaccine is valuable to reduce the course of treatment Bai and colleagues expressed the fusion genes of esat-6 and mpt64 in E.

It was resulted that the more epitopes detected, the stronger immune responses would occur Bao et al. Their results showed that animal challenge would produce specific antibodies In this study, we cloned mpt64 fragment into the eukaryotic pcDNA3. In further studies, immunization can be considered as a DNA vaccine in laboratory animal models. The cells were co-transformed with the pET28 a plasmids coding for the target proteins and the pBirAcm plasmid coding for BirA and induced with 0.

FreeStyle F cells were seeded into fresh FreeStyle expression media with a final density of 1. Then, the mixture was added into 1L FreeStyle F cells. After 2 days, the cells were pelleted at rpm for 5 mins. The pellets were resuspended in 40 ml pre-chilled lysis buffer 20 mM Tris pH 7. The sequences of the plasmids were confirmed by plasmid DNA sequencing.

After each analysis cycle, the sensor chip was regenerated with a buffer containing 6 M guanidine hydrochloride and 0. All measurements were duplicated under the same conditions. The binding affinities K d were determined by fitting the data to a steady-state binding model using Biacore X Evaluation software version 2. Crystals were obtained by mixing the complexes with equal volume reservoir buffer containing 0. The diffraction data were processed with imosflm Three obvious heavy atom peaks were located in the anomalous difference maps calculated with model phases or experimental phases.

The structures were refined using the Phenix package FL-Cas9 expression was verified by western blot analysis. G lentiviral packaging plasmids were gifts from Didier Trono Addgene plasmids and Girds were blotted for 8s and plunge frozen in liquid ethane using a Vitrobot. The dose rate on the camera was set to be Exposure of 8. Images were recorded with a defocus in the range from 1. A total of 3, movies were recorded. The cryo-EM images were subjected to MotionCor2 for whole-frame dose-weighted motion correction.

Particles picking was performed automatically on the summed images in Gautomatch. A total of , particles were picked. Two rounds of 2D classification were then preformed. After discarding bad class averages, , particles were re-centered and re-extracted to Relion 3D classification.

Initial models for reconstruction were generated from scratch by the EMAN2 e2initialmodel. After applying the smoothing procedure for four steps to the original EM map, four regions were obtained.

Phosphorylated residues are underlined and shown in orange. Residues phosphorylated are shown in orange. The data are representative of two independent experiments. The cells were transfected with indicated amounts of pcDNA3. Experiments without cGAMP in the running buffer are indicated. The binding affinity K d was determined by fitting the binding data to a one-site binding model lower panels. The data of b - l are representative of at least two independent experiments.

The frequency of occurrence of an amino acid is indicated underneath the sequence. The cells were stimulated with cGAMP.

The data of b - m , o , and r are representative of at least two independent experiments. The kinase domains KD are in yellow and cyan, the ubiquitin-like domains ULD are in pink and red, the scaffold and dimerization domains SDD are in green and slate. TBK1 dimer is shown by the ribbon representation colored in green and cyan. The TBK1 dimer is shown by the ribbons colored green and cyan.

Mouse TBK1 is shown by the green and cyan cartoon representation. The binding affinity K d was determined by fitting the binding data to a one-site binding model. For each assay, 0. TBK1 knockout cells were transfected with 0. The data of a - h are representative of at least two independent experiments.

The western blot data in j and k are representative of three independent experiments. Indicated amounts of pcDNA3. The data of a - e , l , and m are representative of at least two independent experiments.

The data of f , g , o , and p are representative of at least three independent experiments. Department of Energy under Contract No. We thank Dr. Craig Kaplan of University of Pittsburgh for valuable discussions. Author Information Reprints and permissions information is available at www. The authors declare no competing interests.

Readers are welcome to comment on the online version of the paper. Correspondence and requests for materials should be addressed to P. National Center for Biotechnology Information , U. Author manuscript; available in PMC Nov